Expression of recombinant ribosome inactivating protein MAP30 in E.coli and its biological activity.
- Author:
Li-li ZHANG
1
;
Qian DING
;
Jin-biao ZHAN
Author Information
- Publication Type:Journal Article
- MeSH: Cloning, Molecular; Escherichia coli; genetics; metabolism; Gene Expression; Genetic Vectors; Momordica charantia; chemistry; Recombinant Proteins; biosynthesis; genetics; metabolism; Ribosome Inactivating Proteins, Type 2; biosynthesis; genetics; metabolism; Seeds; chemistry; Transformation, Bacterial
- From: Journal of Zhejiang University. Medical sciences 2010;39(3):264-271
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo clone and produce ribosome inactivating protein MAP30 from the seeds of Momordica charantia L(bitter melon), and to evaluate the biological activity of the recombinant protein.
METHODSThe DNA sequence encoding MAP30 was cloned from the fresh seeds of Momordica charantia by PCR, the target DNA fragments were sequenced after T-A cloning. The expression plasmid was constructed by inserting the MAP30 fragment into vector pET30a. MAP30 was expressed in E.coli by addition of IPTG into final concentration of 1.0 mmol/L. The recombinant MAP30 was identified by SDS-PAGE, and the biological activity of MAP30 protein was evaluated by using MTT assay in cancer cells and normal cells following fluid-phase endocytosis.
RESULTThe nucleotide and amino acid sequences of the cloned MAP30 were identical with those of reported MAP30. The solubility of recombinant protein was analyzed by SDS-PAGE, and the MAP30 was mainly produced in soluble form. The recombinant MAP30 showed a greater cytotoxicity to cancer cells than that to normal cells.
CONCLUSIONThe gene of MAP30 has been successfully cloned.The recombinant MAP30 protein expressed by E.coli is bioactive.
